Process for the preparation of activated polythylene glycols

ABSTRACT

A process for preparing activated polyethylene glycols is disclosed. In some embodiments, the process includes reacting a molten polyethylene glycol with an activator. In other embodiments, the process includes reacting a polyethylene glycol with an activator in the absence of a solvent. The process may be carried out in an inert gas atmosphere, at a temperature at least 10° C. above the melting point of polyethylene glycol, and/or with the activator provided in molar excess of the polyethylene glycol. The invention further provides activated polyethylene glycols produced by this process and their use in a variety of pharmaceutical, medical, cosmetic and chemical applications.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 11/071,877, filed on Mar. 3, 2005, now U.S. Pat. No. 7,351,787,which claims priority to and the benefit of commonly-owned U.S.Provisional Patent Application No. 60/550,817, filed on Mar. 5, 2004,the entire disclosures of each of which are incorporated by referenceherein.

TECHNICAL FIELD

The present invention generally relates to a process for the preparationof activated polyethylene glycols. More specifically, the presentinvention relates to a process for the preparation of activatedpolyethylene glycols without the use of a solvent. The invention furtherprovides activated polyethylene glycols produced by this process andtheir use in a variety of pharmaceutical, medical, cosmetic and chemicalapplications.

BACKGROUND

Polyethylene glycol (PEG) is a polymer having the structureH(O—CH₂—CH₂)_(n)—OH. It is generally synthesized by the ring-openingpolymerization of ethylene oxide. PEGs of different molecular weighthave previously been used in a number of biomedical applications,including processes known as “pegylation,” in which PEG is attached to aprotein to extend its activity.

To effect covalent attachment of polyethylene glycol to a protein, thehydroxyl end-groups of the polymer need to be converted to reactivefunctional groups. This process is frequently referred to as“activation” and the product is called “activated PEG.” Followingactivation, the reactive functional end-groups of the activated PEG canthen react with various functional groups on a variety of molecules,e.g., proteins.

Several chemical procedures have been developed to prepare activatedPEGs. For example, PEGs have been successfully activated by reactionwith 1,1-carbonyl-di-imidazole, cyanuric chloride, tresyl chloride,2,4,5-trichlorophenyl chloroformate, 4-nitrophenyl chloroformate, andvarious N-hydroxy-succinimide derivatives, as well as by theMoffatt-Swern reaction. See Beauchamp et al., Anal. Biochem., 131: 25(1983); Nashimura et al., Life Sci., 33: 1467 (1983); Delgado et al.,Appl. Biochem., 12: 119 (1990); Wirth et al., Bioorg. Chem., 19: 133,(1991); Veronese et al., Biochem. Biotechnol., 11: 141 (1985); Sartoreet al., Biochem. Biotechnol., 27: 45 (1991); Anderson et al., J.Immunol. Methods, 109: 37 (1988); and Zalipsky et al., J. Bioact.Compat. Polym., 5: 227 (1990).

The activation of PEGs with 4-nitrophenyl chloroformate to generatePEG-dinitrophenyl carbonates has been described by Fortier andLaliberte. See Fortier et al., Biotech. Appl. Biochem., 17: 115 (1993).The reaction was carried out in acetonitrile containing triethylamine(TEA) for 5 hours at 60° C. To keep an anhydrous environment during thereaction, it was necessary to use a cumbersome Soxhlet extractionsystem.

International Publication No. WO 03/018665 describes an alternativemethod for preparing activated PEGs. The method involves a reactioncarried out at room temperature using an aprotic solvent, such asmethylene chloride (CH₂Cl₂), in the presence of a catalyst, such asdimethylaminopyridine (DMAP). The use of solvents in both the reactionitself and the subsequent extraction steps to remove by-products, suchas the toxic hydrochloric acid salt of DMAP, increases the cost of thefinal product.

Thus there is a need to develop an efficient, low-cost, andenvironmentally friendly method for preparing activated PEGs that willminimize or eliminate the use of solvents, catalysts, toxic compounds,cumbersome equipment, and/or purification steps.

SUMMARY OF THE INVENTION

It has been discovered that activated polyethylene glycols can beprepared via a process that eliminates or minimizes the use of solvents,catalysts, toxic compounds, cumbersome equipment, and/or purificationsteps. In addition, the process can prepare polyethylene glycols with ahigh degree of activation. Generally, the invention is a process forpreparing activated polyethylene glycols that includes reacting apolyethylene glycol with an activator, which transfers a leaving groupto the polyethylene glycol. The process is conducted under solvent-freeconditions such that the polyethylene glycol serves as the reactionmedium, e.g., the polyethylene glycol is in a molten or liquid state.

According to one aspect of the invention, a process for preparing anactivated polyethylene glycol may include the steps of providing amolten polyethylene glycol and reacting the molten polyethylene glycolwith an activator. The molten polyethylene glycol may have a generalformula of M-(OCH₂CH₂)_(n)—O-M, wherein n may be an integer greater than2, and M may be selected from the group consisting of H, Li, Na, K, Rb,and Cs. The activator may have a general formula of Y-Q-X, wherein X andY independently may be a leaving group, and Q may be selected from thegroup consisting of —C(O)—, —SO₂—, >P(O)—, and -A-R-B-, wherein A and Bindependently may be —C(O)—, —SO₂—, or >P(O)—, and R may be selectedfrom the group consisting of a lower alkyl group, a lower branched alkylgroup, an aryl group, an aralkyl group, —CHR₁)_(t)—, —O—(CHR₁)_(t)—O—,—S—(CHR₁)_(t)—S—, —O—(CHR₁)_(t)—S—, —S—(CHR₁)_(t)—O—,—O—(CHR₁)_(t)—R₂C═CR₃—(CHR₄)_(t)—O—,—S—(CHR₁)_(t)—R₂C═CR₃—(CHR₄)_(t)—S—,—O—(CHR₁)_(t)—R₂C═CR₃—(CHR₄)_(t)—S—, and—S—(CHR₁)_(t)—R₂C═CR₃—(CHR₄)_(t)—O—. The variables R₁, R₂, R₃, and R₄may be independently selected from the group consisting of a hydrogenatom, a lower alkyl group, a lower branched alkyl group, an aryl group,and an aralkyl group, and t may be 1, 2, or 3. The activatedpolyethylene glycol prepared according to this aspect of the inventionmay have a general formula of Y-Q-(OCH₂CH₂)_(n)—O-Q-Y, wherein Q, Y, andn are as defined hereinabove.

In some embodiments, X and Y may be independently selected from thegroup consisting of a halide group, a mesyl group, a tosyl group, aphenoxyl group, and a substituted phenoxyl group. The activator mayinclude, but is not limited to, ClSO₂Cl, ClCOCH₂SO₂Cl, 4-O₂NPhOCOCl,2-O₂NPhOCOCl, PhOCOCl, ClSO₂CH₂CH₂SO₂Cl, POCl₃, PhOPOCl₂, PhPOCl₂,CCl₃COCl, and CBr₃COCl. In preferred embodiments, the activator may beO₂NPhOCOCl.

In some embodiments, the reacting step may be carried out at atemperature at least about 10° C. above the melting point of thepolyethylene glycol. For example, for a polyethylene glycol having theformula M-(OCH₂CH₂)_(n)—O-M wherein M is H and n is an integer between150 and 250, the reacting step may be carried out at a temperature inthe range of about 60-90° C., preferably in the range of about 70-80°C., and more preferably at about 75° C.

The reacting step may be carried out in an atmosphere that allows theeffective removal of gaseous products formed during the reaction. Insome embodiments, the reacting step may be carried out in an inert gasatmosphere, e.g., under nitrogen. In alternative embodiments, thereacting step may be carried out under vacuum.

The activator may be added in a stepwise manner. In some embodiments,the process may include reacting the polyethylene glycol with two ormore portions of the activator. For example, two or more portions of theactivator comprising 1-199% of the equimolar amount of the polyethyleneglycol may be added.

In other embodiments, the process may include reacting the polyethyleneglycol with an excess of the activator. For example, the molar amount ofthe activator may be in 50-100% excess of the molar amount of thepolyethylene glycol. In preferred embodiments, the activator may beprovided in 25-75% molar excess. In particular embodiments, theactivator may be provided in 33-66% molar excess.

According to another aspect of the invention, a process for preparing anactivated polyethylene glycol may include the steps of providing apolyethylene glycol and reacting the polyethylene glycol with anactivator in the absence of a solvent. The polyethylene glycol may havea general formula of M-(OCH₂CH₂)_(n)—O-M, while the activator may have ageneral formula of Y-Q-X. The activated polyethylene glycol preparedaccording to this aspect of the invention may have a general formula ofY-Q-(OCH₂CH₂)_(n)—O-Q-Y. The variables M, Q, X, Y, and n are as definedhereinabove.

Activated polyethylene glycols prepared by the processes described aboveare within the scope of the present invention. In one aspect, theactivated polyethylene glycols prepared by the processes of theinvention may be reacted with a molecule including a functional groupthat reacts with the activated polyethylene glycol to form a covalentbond. In some embodiments, the molecule may be a protein, preferably, analbumin. For example, the activated polyethylene glycols prepared by theprocesses of the present invention may be used to form hydrogels bymixing the activated polyethylene glycols with proteins dissolved inaqueous solutions. In some embodiments, a backing may be applied to thehydrogel to form a medical article, e.g., a wound dressing, adapted tobe applied to a mammal such as a human. The activated polyethyleneglycols also may be used as linkers for resins, and they may be readilylinked to proteins or reacted with enzyme surfaces.

The foregoing, and other features and advantages of the invention aswell as the invention itself, will be more fully understood from thefollowing figures, description, and claims.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 is a representative ¹H NMR spectrum of PEG-8000 that has beentrifluoroacylated.

FIG. 2 is a representative ¹H NMR spectrum of PEG-NPC₂ preparedaccording to an embodiment of the invention.

FIG. 3 is a representative ¹H NMR spectrum of p-nitrophenol.

FIG. 4 shows activation degree as a function of reaction time forPEG-NPC₂ prepared according to an embodiment of the invention asdetermined by NMR and SEC. Results from two separate sets of NMRcalculations are presented. One set of NMR calculations were performedwith the aromatic proton signals at 7.40 and 8.25 ppm. A second set ofNMR calculations were performed with the methylene end-group protonsignal at 4.48 ppm.

FIGS. 5A-5C are representative ¹H NMR spectra of PEG-NPC₂ preparedaccording to an embodiment of the invention after (a) 1 hour, (b) 90minutes, and (c) 3 hours of reaction time. Increasing signals at 7.45and 8.25 ppm attributed to unknown impurities can be observed.

FIG. 6 shows size-exclusion chromatograms of PEG-NPC₂ prepared accordingto an embodiment of the invention after (a) 45 minutes, (b) 60 minutes,(c) 90 minutes, and (d) 180 minutes of reaction time.

FIG. 7 shows melting point as a function of activation degree ofPEG-NPC₂ prepared according to an embodiment of the invention and analternative process.

FIG. 8 shows the efficiency of activated carbon sorption ofp-nitrophenol in aqueous solutions of activated PEG with a polymerconcentration of 22% (w/v).

FIG. 9 shows the correlation between the adsorption capacity ofactivated carbon and the concentration of p-nitrophenol at equilibrium.

FIG. 10 shows specific sorption as a function of weight ratio withregard to the adsorption of p-nitrophenol by activated carbon in aqueoussolutions of activated PEG with a polymer concentration of 22% (w/v).

DETAILED DESCRIPTION

It has been discovered that activated polyethylene glycols can beprepared under solvent-free conditions. In some embodiments, the processmay include reacting a molten polyethylene glycol with an activator. Inother embodiments, the process may include reacting a polyethyleneglycol with an activator in the absence of a solvent. The process may becarried out in an inert gas atmosphere, at a temperature at least 10° C.above the melting point of polyethylene glycol, and/or with theactivator provided in molar excess of the polyethylene glycol. Theinvention further provides activated polyethylene glycols produced bythis process and their use in a variety of pharmaceutical, medical,cosmetic and chemical applications.

As used herein, the term “lower alkyl” group refers to “C₁₋₂ alkyl”,“C₁₋₃ alkyl”, “C₁₋₄ alkyl”, “C₁₋₅ alkyl”, “C₁₋₆ alkyl”, i.e., an alkylgroup having one to two, one to three, one to four, one to five, one tosix carbon atoms, respectively such as, for example, methyl, ethyl,propyl, butyl, pentyl, hexyl, and their isomeric forms thereof. Theisomeric forms include “lower branched alkyl” groups as known in theart.

The term “halide” group refers to a charged or uncharged fluorine atom,a chlorine atom, a bromine atom, and/or an iodine atom.

The term “aryl” group refers to a mono- or bicyclic carbocyclic ringsystem having one or two aromatic rings including, but not limited to,phenyl, naphthyl, tetrahydronaphthyl, indanyl, idenyl and the like.

The term “substituted aryl” group refers to an aryl group, as definedherein, substituted by independent replacement of one, two, three, four,or five of the hydrogen atoms thereon with substituents independentlyselected from alkyl, substituted alkyl, haloalkyl, alkoxy, thioalkoxy,amino, alkylamino, dialkylamino, acylamino, cyano, hydroxy, halo,mercapto, nitro, carboxaldehyde, carboxy, alkoxycarbonyl andcarboxamide. More specifically, the substituents may be F, Cl, Br, I,OH, NO₂, CN, C(O)—C₁₋₆ alkyl, C(O)-aryl, C(O)-heteroaryl, CO₂-alkyl,CO₂-aryl, CO₂-heteroaryl, C(O)NH₂, C(O)NH—C₁₋₆ alkyl, C(O)NH-aryl,C(O)NH-heteroaryl, OC(O)—C₁₋₆ alkyl, OC(O)-aryl, OC(O)-heteroaryl,OCO₂-alkyl, OCO₂-aryl, OCO₂-heteroaryl, OC(O)NH₂, OC(O)NH—C₁₋₆ alkyl,OC(O)NH-aryl, OC(O)NH-heteroaryl, NHC(O)—C₁₋₆ alkyl, NHC(O)-aryl,NHC(O)-heteroaryl, NHCO₂-alkyl, NHCO₂-aryl, NHCO₂-heteroaryl, NHC(O)NH₂,NHC(O)NH—C₁₋₆ alkyl, NHC(O)NH-aryl, NHC(O)NH-heteroaryl, SO₂—C₁₋₆ alkyl,SO₂-aryl, SO₂-heteroaryl, SO₂NH₂, SO₂NH—C₁₋₆ alkyl, SO₂NH-aryl,SO₂NH-heteroaryl, C₁₋₆ alkyl, C₃₋₆ cycloalkyl, CF₃, CH₂CF₃, CHCl₂,CH₂OH, CH₂CH₂OH, CH₂NH₂, CH₂SO₂CH₃, aryl, heteroaryl, benzyl, benzyloxy,aryloxy, heteroaryloxy, C₁₋₆ alkoxy, methoxymethoxy, methoxyethoxy,amino, benzylamino, arylamino, heteroarylamino, C₁₋₃ alkylamino, thio,aryl-thio, heteroarylthio, benzyl-thio, C₁₋₆ alkyl-thio, ormethylthiomethyl. In addition, substituted aryl groups includetetrafluorophenyl and pentafluorophenyl.

The term “aralkyl” group refers to an aryl group attached to an alkylgroup. An example of an arylalkyl group is a benzyl group.

The term “substituted aralkyl” group refers to an aryl group orsubstituted aryl group attached to an alkyl group or a substituted alkylgroup, provided that one or both of the aryl and alkyl groups aresubstituted.

The term “aroxyl” group refers to an aryl group attached to an oxygenatom. An example of an aroxyl group is a phenoxyl group.

The term “substituted aroxyl” group refers to an aroxyl group or asubstituted aroxyl group attached to an alkyl group or a substitutedalkyl group, provided that one or both of the aroxyl and alkyl groupsare substituted.

Throughout the description, where compositions are described as having,including, or comprising specific components, or where processes aredescribed as having, including, or comprising specific process steps, itis contemplated that compositions of the present invention also consistessentially of, or consist of, the recited components, and that theprocesses of the present invention also consist essentially of, orconsist of, the recited processing steps.

It should be understood that the order of steps or order for performingcertain actions is immaterial so long as the invention remains operable.Moreover, two or more steps or actions may be conducted simultaneously.

The PEG activation process of the invention may be described by thegeneral reaction:M-(O—CH₂—CH₂)_(n)—OM+2Y-Q-X→Y-Q-(OCH₂CH₂)_(n)—O-Q-Y+2MX  (Equation 1)

The unmodified polyethylene glycol has the formula M-(O—CH₂CH₂)_(n)—OMwherein n is an integer greater than 2, and M is selected from the groupconsisting of H, Li, Na, K, Rb, Cs, and Fr. In preferred embodiments, Mmay be a hydrogen atom, and M-(O—CH₂—CH₂)_(n)—OM may be a polyethyleneoxide. In certain embodiments, n may be an integer between 4 and 800. Inparticular embodiments, n may be an integer between 65 and 800, i.e.,the unmodified PEG may have a molecular weight of about 3,000 Da to35,000 Da. In preferred embodiments, n may be an integer between 150 and250, corresponding to unmodified PEGs of molecular weights between 6000and 11,000 Da.

The polyethylene glycol may be reacted with an activator of the formulaY-Q-X. X and Y independently may be a leaving group. A leaving group isan atom or group (charged or uncharged) that may be displaced from anatom in which is considered the main part of the compound. For example,a leaving group may be an anion or a neutral molecule. Leaving groupssuitable for activating polyethylene glycols as well as activators foruse in methods of the invention are well known in the art and includeprotective groups commonly used in organic synthesis. See, e.g., J. W.Barton (1973) PROTECTIVE GROUPS IN ORGANIC CHEMISTRY, Chapter 2, PlenumPress, New York.; T. W. Greene and P. G. M. Wuts (1999) PROTECTIVEGROUPS IN ORGANIC SYNTHESIS, 3rd edition, John Wiley & Sons, Inc., NewYork. In preferred embodiments, X and Y may be selected from the groupconsisting of a halide group, a mesyl group, a tosyl group, an aroxylgroup such as a phenoxyl group, and a substituted aroxyl group such as asubstituted phenoxyl group.

In accordance with the formula Y-Q-X, Q may be selected from the groupconsisting of —SO₂—, —C(O)—, >P(O)—, and -A-R-B-, wherein A and Bindependently may be —SO₂—, —C(O)—, or >P(O)—, and R may be selectedfrom the group consisting of a lower alkyl group, a branched lower alkylgroup, an aryl group, an aralkyl group, —(CHR₁)_(t)—, —O—(CHR₁)_(t)—O—,—S—(CHR₁)_(t)—S—, —O—(CHR₁)_(t)—S—, —S—(CHR₁)_(t)—O—,—O—(CHR₁)_(t)—R₂C═CR₃—(CHR₄)_(t)—O—,—S—(CHR₁)_(t)—R₂C═CR₃—(CHR₄)_(t)—S—, —O—(CHR₁)_(t)R₂C═CR₃—(CHR₄)_(t)—S—,and —S—(CHR₁)_(t)—R₂C═CR₃—(CHR₄)_(t)—O—C(O)(CHR₁)_(t)SO₂—, wherein t maybe an integer between 1 and 3, and R₁, R₂, R₃ and R₄ independently maybe selected from the group consisting of a hydrogen group, a lower alkylgroup, a branched lower alkyl group, an aryl group, and an aralkylgroup. In some embodiments, R₁, R₂, R₃ and R₄ may be selected from thegroup consisting of a hydrogen atom, a methyl group, an ethyl group, anisopropyl group, and a propyl group. In preferred embodiments, Q may be—SO₂—, —C(O)—O—CH₂—CH═CH—CH₂—O—C(O)—, —C(O)CH₂SO₂—, —C(O)—, and—SO₂CH₂CH₂SO₂—. In some embodiments, Q may have the formula:Q=(U₀)n₀-[(Q₀)n₁(CHR₁)n₂-(Q₁)n₃]_(p)-[(R₂C═CR₃)n₄-(Q₂)n₅]_(m)-[(CHR₄)n₆-(Q₃)n₇]_(q)-(U₁)n₈  (Formula1)wherein Q₀, Q₁, Q₂, and Q₃ independently may be O or S; U₀ and U₁ may beindependently selected from the group consisting of —C(O)—, —SO₂—,and >P(O)—; n₀, n₁, n₃, n₄, n₅, n₇, and n₈ independently may be 0 or 1;n₂ and n₆ independently may be 0, 1, 2, or 3; p and q independently maybe 0, 1, 2, 3, 4, 5; or 6; and m may be 0, 1, or 2, wherein 0≦p+m≦8. Thevariables R₁, R₂, R₃, and R₄ are as defined hereinabove.

The activator Y-Q-X may be capable of the in situ generation of ionsincluding, but not limited to, acylium, sulfonium, sulfurylium andphosphonium ions.

Selected examples of preferred activators include, but are not limitedto:

Cl—C(O)—OPhNO₂, wherein, according to Formula 1, n₀=1; n₁=n₂=n₃=n₈=0;p=1; m=q=0, Q=U₀=—C(O)—, and wherein X=Cl and Y=—OPhNO₂;

Cl—SO₂OCH₂CH₂OSO₂—Cl, wherein, according to Formula 1, n₀=n₁=n₃=n₈=1;n₂=2; p=1; m=q=0; U₀=U₁=SO₂; Q₀=Q₁=O (oxygen); R₁=H, Q=—SO₂OCH₂CH₂OSO₂—and wherein X═Y═Cl;

Cl—SO₂OCH₂CH₂OC(O)—Cl, wherein, according to Formula 1,n₀=n₁=n₂=n₆=n₇=n₈=1; n₃=0; p=q=1; m=0; U₀=SO₂; U₁=—C(O)—; Q₀=Q₃=O(oxygen); R₁=R₄=H, Q=—SO₂OCH₂CH₂OC(O)—, and wherein X═Y═Cl; and

Cl—C(O)OCH₂CH═CHCH₂OC(O)—Cl, wherein, according to Formula 1,n₀=n₁=n₂=n₄=n₆=n₇=n₈=1; n₃=n₅=0; p=m=q=1; U₀=U₁=—C(O)—; Q₀=Q₃=O(oxygen); R₁=R₂=R₃=R₄=H, Q=—C(O)OCH₂CH═CHCH₂OC(O)—, and wherein X═Y═Cl.

Other activators may be selected from, but are not limited to, ClSO₂Cl,ClC(O)CH₂SO₂Cl, 4-O₂NPhOC(O)Cl, 2-O₂NPhOC(O)Cl, PhOC(O)Cl,ClSO₂CH₂CH₂SO₂Cl, P(O)Cl₃, PhOP(O)Cl₂, PhP(O)Cl₂, CCl₃C(O)Cl, andCBr₃C(O)Cl.

Selected examples of bis-activated PEGs having the general formula:Y-Q-(OCH₂CH₂)_(n)—O-Q-Y  (Formula 2)include, but are not limited to:

wherein Y is Cl, Q is —SO₂—, and n is an integer between 4 and 800, i.e.a PEG having a molecular weight from about 200 Da to 35,000 Da;

wherein Y is Cl, Q is —C(O)CH₂SO₂—, and n is an integer between 4 and800, i.e. a PEG having a molecular weight from about 200 Da to 35,000Da;

wherein Y is Cl, Q is —SO₂CH₂CH₂SO₂—, and n is an integer between 4 and800, i.e. a PEG having a molecular weight from about 200 Da to 35,000Da; and

wherein Y is 4-O₂NPhO—, Q is —C(O)—, and n is an integer between 4 and800, i.e. a PEG having a molecular weight from about 200 Da to 35,000Da.

In some embodiments of the present invention, the activation process mayinclude reacting a molten polyethylene glycol with an activator,preferably having the formula Y-Q-X. The activation process may takeplace in the absence of a solvent.

A molten polyethylene glycol may be provided by heating an unmodifiedpolyethylene glycol to or above its melting point. For example, theunmodified polyethylene glycol may be heated at a temperature range inwhich all, substantially all, or a significant proportion (for example,more than about 30% by weight, more than about 40% by weight, more thanabout 50% by weight, more than about 60% by weight, more than about 70%by weight, more than about 80% by weight, more than about 90% by weight,or more than about 95% by weight, more than about 30% by volume, morethan about 40% by volume, more than about 50% by volume, more than about60% by volume, more than about 70% by volume, more than about 80% byvolume, more than about 90% by volume, or more than about 95% by volume)of the polyethylene glycol is melted. The corresponding temperaturerange may be, for example, from about 50° C. to about 80° C.

The reacting step may be carried out at a temperature at least about 10°C. above the melting point of the unmodified polyethylene glycol. For anunmodified PEG having a molecular weight of 8 kDa, the reacting step maybe carried out at a temperature in the range of 60-90° C., preferably inthe range of 70-80° C., and more preferably at 75° C.

Because hydrochloric gases or other by-products may be produced over thecourse of the reaction, the reacting step may be carried out in anatmosphere that allows the effective removal of the gaseous productsformed during the reaction. In certain embodiments, the reacting stepmay be carried out in an inert gas atmosphere. For example, the reactingstep may be carried out under nitrogen. In alternative embodiments, thereacting step may be carried out under vacuum.

Reaction between the unmodified polyethylene glycol and the activatormay be initiated by adding a desired amount of the activator to themolten polyethylene glycol. In some embodiments, the entire amount ofthe activator may be added at the beginning of the reaction. In otherembodiments, the activator may be added in a stepwise manner. Forexample, the activator may be added in two or more separate portions.Each separate portion may comprise 1-199% of the equimolar amount of thepolyethylene glycol. In preferred embodiments, portions of the activatorcomprising about 20-60% of the equimolar amount of PEG may be added atselected time intervals. For example, portions of the activator may beadded at about 1-60 minute interval(s), preferably, at about 10-40minute interval(s), and more preferably, at about 15-minute intervals.

It was found that longer reaction times, for example two hours or more,may produce undesirable side reactions accompanied by the formation ofan increased level of impurities. Accordingly, in certain embodiments,the reaction may be allowed to occur no longer than 120 minutes from thefirst addition of the activator. In preferred embodiments, the reactionmay be allowed to occur no longer than 90 minutes from the firstaddition of the activator.

Additionally, it was found that a molar excess of the activator may benecessary to completely activate, or substantially completely activate,the unmodified polyethylene glycol. An activation degree of 100%indicates that a polyethylene glycol is completely activated. Asubstantially completely activated PEG may have an activation degree of,for example, 90-99%. In certain embodiments of the present invention,the activator may be provided in molar excess to the polyethyleneglycol. For example, the molar amount of the activator may be in 100%excess of the molar amount of the polyethylene glycol. In preferredembodiments, the activator may be provided in 25-75% excess of the molaramount of the polyethylene glycol. In more preferred embodiments, theactivator may be provided in 33-66% excess of the molar amount of thepolyethylene glycol.

The use of excess reagents may influence the purification process of thefinal product. For example, it was found that free p-nitrophenols (pNP)and bis-(p-nitrophenyl carbonate)s may be present as impurities and/orby-products in the reaction system when the activator is nitrophenylchloroformate (NPCF). Because even a small amount of these impuritiesare known to inhibit desirable reactions between activated PEG andproteins, the process of the invention may be followed by a purificationstep including the addition of activated carbon (AC) to adsorb theseimpurities. It was found that an approximately 10-20% weight ratio ofAC:PEG may be effective in removing 90-95% of these impurities producedduring the reaction. The use of activated carbon as a purificationtechnique for the activated PEG produced by the invention is simple,cost-effective, and ecologically friendly. For example, activated carbonthat has been used to purify the activated PEGs of this invention can beeasily reactivated and recycled. Alternatively or complementarily,elimination of free p-nitrophenols can be achieved through deionizationof the bulk activated PEG solution by means of ion-exchange technique.Anion-exchange resins in the free hydroxyl form (such as Amberlite® IRA67 available from Sigma-Aldrich) are particularly suitable forabsorption of p-nitrophenol from aqueous solutions of activated PEG.After separation via filtration or centrifugation, the anion-exchangematerial with absorbed impurities can be recycled and used again. Theactivated polyethylene glycol provided by the process of the inventionmay be further treated by spray-drying or lyophilization technique toimprove stability and to prolong shelf life.

Activated polyethylene glycols produced by the invention may be reactedwith a molecule including a functional group that reacts with theactivated polyethylene glycol to form a covalent bond. Examples of sucha functional group include, but are not limited to, hydroxyl groups,amine groups and thiol groups. The functional group may be derived fromor forms part of a peptide, a protein, a saccharide, a polysaccharide,and/or an oligonucleotide. The formation of the covalent bond may resultin the formation of a bio-polymer, which may be used in chemical, food,cosmetical, cosmeceutical, pharmaceutical, and dermopharmaceuticalapplications.

For example, activated polyethylene glycols prepared by the process ofthe invention may be used to form hydrogels by mixing the activated PEGwith proteins dissolved in aqueous solutions. The activated PEG may bemixed with a protein (such as albumin, casein, hydrolyzed soy protein,or other animal-based or vegetal proteins) in an aqueous solution underbasic conditions to form a three-dimensional reticulated mixture orhydrogel. See, e.g., International Publication No. WO 01/74928. Inparticular embodiments, the three-dimensional reticulated mixture orhydrogel may be attached to a backing, e.g., a polymer backing, toprovide a medical article, such as an active ingredient delivery device,a wound cover/dressing, or a diagnostic tool. The hydrogel may beattached to the backing with or without the use of adhesives. Forexample, a polymer backing may be made adhesive to the hydrogel byexposing the surface of the polymer backing to an activated gas, i.e., aplasma of various gases or mixtures of gases produced by an excitationsource such as microwave and radiofrequency, as described inInternational Publication No. WO 02/070590. Activated PEGs preparedaccording to the present invention also may be used as linkers forresins, and they can be readily linked to proteins or reacted withenzyme surfaces.

The following examples are provided to illustrate further and tofacilitate the understanding of the invention and are not intended tolimit the invention.

EXAMPLE 1 Preparation of Polyethylene Glycol Dinitrophenyl Carbonate(PEG-NPC₂) Via Solvent-free, One-time Addition Method

Polyethylene glycol of molecular weight 8000 Da (PEG-8000) (FischerScientific, 300.0 g, 37.5 mmol) was placed in a vacuum flask equippedwith a thermometer and a stirrer. Upon heating to about 65° C. to 70°C., the PEG powder began to melt. Once the PEG powder was completelymelted, p-nitrophenyl chloroformate (p-NPCF) (ABCR GmbH & Co. KG,Karlsruhe, Germany, 16.6 g, 82.5 mmol, 10% molar excess) was added tothe molten PEG to form a viscous solution. The reaction mixture wasstirred at about 70° C. to 75° C. for three hours under vacuum ornitrogen, after which the mixture was poured onto a plastic plate tocool. The PEG-NPC₂ product was ground into a powder and storedrefrigerated.

EXAMPLE 2 Preparation of PEG-NPC₂ Via Solvent-free, Stepwise-additionMethod

PEG-NPC₂ was prepared similar to the method described in Example 1,except that the activator p-NPCF was added in portions. Polyethyleneglycol of molecular weight 8000 Da (PEG-8000) (Fischer Scientific, 300.0g, 37.5 mmol) was placed in a vacuum flask equipped with a thermometerand a stirrer. Upon heating to about 65° C. to 70° C., the PEG powderbegan to melt. Once the PEG powder was completely melted, portions ofp-NPCF comprising 33% of the equimolar amount of the terminal OH groupsof PEG were added to the molten PEG at 15-minute intervals until a 200%molar excess of p-NPCF was added in total. Table 1 provides the specificamount of p-NPCF added over the course of the reaction. The reactionmixture was continuously stirred for two hours at about 70° C. to 75°C., then kept under vacuum overnight to remove residual HCl vapors. Thecrystallized PEG-NPC₂ product was ground into a powder and keptrefrigerated.

TABLE 1 Amount of p-NPCF added in grams per 300 grams of PEG over time.Amount of Molar ratio to OH Time (min) p-NPCF added (g) groups of PEG(%) 0 0.0 0 15 5.0 33 30 10.0 67 45 15.0 100 60 20.0 133 75 25.0 167 9030.0 200 105 35.0 233

EXAMPLE 3 Preparation of PEG-NPC₂ Via In-solvent Method

PEG-8000 (Fischer Scientific, 363.4 g, 45.0 mmol) was dissolved inanhydrous CH₂Cl₂ (500 mL). p-NPCF (19.63 g, 97.4 mmol) dissolved inCH₂Cl₂ (50 mL) was added to the PEG solution in a 8.0 L reaction vesselupon vigorous stirring. A solution prepared with 12.2 g of DMAPdissolved in 50 ml of anhydrous CH₂Cl₂ was added to the mixture whilestirring continued. The reaction mixture was kept at room temperatureand stirred for another 2 hours.

The first purification step involved precipitation with cold diethylether (2.0 L, 4° C.). The resulting suspension was cooled in arefrigerator set at −20° C. for 30 minutes. The suspension was filteredunder vacuum, and the precipitate was washed several times with colddiethyl ether. The washed precipitate was re-suspended in water, stirredvigorously for about 30 minutes, and filtered under vacuum again. Theresulting yellowish filtrate was extracted with dichloromethane threetimes. The combined solvent fractions were filtered over Na₂SO₄ andconcentrated. The resulting product was precipitated in cold diethylether upon vigorous stirring. The PEG-NPC₂ product obtained wasfiltered, washed with diethyl ether, and dried under vacuum.

EXAMPLE 4 Determining Activation (Substitution) Degree of PEG-NPC₂

A. Nuclear Magnetic Resonance

To better understand the peculiarities of the reaction, a kinetic studywas conducted with activated PEG samples prepared with differentstoichiometric ratios of PEG/NPCF according to the procedures describedin Example 2 and Table 1. Specifically, the activation degree of thesesamples was measured by means of nuclear magnetic resonance spectroscopy(NMR).

¹H NMR spectroscopy was performed on a 300 MHz Varian type NMRinstrument. Samples were placed in 5 mm NMR tubes dissolved indeuterated chloroform (CDCl₃). For all polymer samples, 128 scans wereselected.

In this NMR study, the ¹H NMR spectra of unmodified PEG-8000 and theintegrated values of proton peaks were taken as the standard for furthercalculations. The signal from the hydroxyl end-groups of the unmodifiedPEG, however, was not resolved. To observe the signal produced by thesehydroxyl groups, trifluoroacetic anhydride was added 3 hours prior tomeasurements. Trifluoroacetic anhydride is known to substitute hydroxylgroups with 100% efficiency. See Jo et al., Biomacromol., 2: 255-261(2001); Tessmar et al., Biomacromol., 3: 194-200 (2002); Behravesh etal., Biomacromol., 3: 153-158 (2002).

FIGS. 1 and 2 are representative NMR spectra of trifluoroacylated PEGand PEG-NPC₂, respectively. FIG. 3 is a representative NMR spectrum ofp-nitrophenol (pNP). Signals observed in the NMR spectrum of PEG-NPC₂(FIG. 2) were assigned by comparison with the spectra oftrifluoroacylated PEG (FIG. 1) and pNP (FIG. 3). The assignment of theNMR signals of PEG-NPC₂ is provided in Table 2.

TABLE 2 Assignment of NMR signals of PEG-NPC₂. Signal (ppm) Associatedchemical structure ~1.98-2.00 Residual solvents and reactants e.g., DMAPand CH₂Cl₂ (observed only in PEG-NPC₂ samples prepared according to thein-solvent method) ~3.6-3.8 PEG structure associated protons(—O—CH₂—CH₂—O)_(n) ~4.48-4.50 Protons from methylene groups adjacent tosubstitution NPC—(O—CH₂—CH₂—O—)_(n) Duplet at ~6.95 and 8.10 Free pNPmolecules ~7.25 Solvent for measurement (chloroform) Duplet at ~7.40 and8.25 NPC substitution Duplet at ~7.45 and 8.25 Impurities, presumablybis(p-nitro- phenyl carbonate)s (observed only in PEG-NPC₂ samplesprepared according to the solvent-free method)

The substitution of the OH groups in the PEG-NPC₂ samples was confirmedby the singlet at 4.48 ppm. The singlet, which appears in the spectra ofboth trifluoroacylated PEG (FIG. 1) and PEG-NPC₂ (FIG. 2), was assignedto the two ethylene protons adjacent to the substituted groups. Thesignals at 6.95 and 8.10 ppm were assigned to free p-nitrophenolmolecules produced as a by-product of the reaction. The same signals canbe observed in the NMR spectrum of pNP (FIG. 3).

The ratio of the four protons of the two terminal methylene groups at4.48-4.50 ppm to the main chain protons at 3.6-3.8 ppm was calculatedfrom three separate spectra obtained with trifluoroacylated PEG. Theestimated ratio is 4 to 820 (±5% of error). This is comparable to thetheoretical ratio of 4 to 728, which was calculated as follows. Becausethe repeating unit of PEG has a formula molecular weight of 44, thepolymerization degree (n) of a PEG macromolecule of molecular weight8000 is about 182. Since the repeating unit comprises 4 protons, thetotal number of protons in the main chain is about 728 (4×182).Discrepancy in the total proton content may be attributed to smalldeviations in the molecular weight values from the nominal value 8000.

Based on this integral ratio of 4 to 820 (or 1 to 205) obtained from100% trifluorosubstituted PEG, the activation degree of differentPEG-NPC₂ samples was determined over the course of their synthesis.

The results are summarized in FIG. 4. It was observed that the PEGsamples were fully activated (reaching an activation degree of 100%) asearly as about 105 minutes into the reaction, which corresponds to theaddition of 133% molar excess of p-NPCF. The NMR data also suggest thatlonger reaction time, i.e., more than 2 hours, could lead to undesirableside reactions and the formation of impurities, such asbis(p-nitrophenyl carbonate)s, rather than a higher activation degree.

The increase in impurities level over longer reaction times can be seenmore clearly in FIGS. 5A-5C, which are representative NMR spectra ofPEG-NPC₂ samples prepared according to the solvent-free method describedin Example 2 taken after (a) 1 hour, (b) 90 minutes, and (c) 3 hours ofreaction time. The signal at 7.45 and 8.25 ppm, which is assigned tounknown impurities content, was observed to increase (from FIG. 5A toFIG. 5C) over longer reaction times.

To compare the effect of the different preparation methods on the degreeof activation of an activated PEG sample, spectra of PEG-NPC₂ samplesprepared by each of the methods of Examples 1, 2 and 3 were obtained.Specifically, the integral ratio of the signals at 7.40 and 8.25 ppmattributed to the aromatic protons from the nitrophenyl substituents wasused to calculate the activation degree of PEG-NPC₂ over time. It wasrecognized that the appearance of the impurities signals at 7.45 and8.25 ppm might preclude accurate measurements. To verify the accuracy ofthe calculations with the aromatic protons, a second set of calculationswere performed with the methylene end-group proton signals at 4.48 ppm.The results are summarized in Table 3.

TABLE 3 Activation degree (AD) of activated PEG samples prepared by (i)the solvent-free, one-time addition method (Example 1), (ii) thesolvent-free, stepwise addition method (Example 2), and (iii) thein-solvent method (Example 3). The sample indicated by * was kept undervacuum overnight after the reaction. Samples indicated by ** wererecrystallized in acetonitrile. Amount of AD Calculated AD CalculatedReaction p-NCPF with Signals at with Signal Preparation Time added 7.40and 8.25 at 4.48 ppm Method (min) (mol %) ppm (%) (%) Example 1 60 11063 37 Solvent-free, 120  110 65 one-time **120   110 63 76 addition 180 110 64 58 method **180   110 69 73 Example 2 45 100 71 50-60Solvent-free, 60 133 84 70 stepwise- 75 167 96 76 addition 90 200 100 88method 105  233 100 100  180  300 100 95-98 *90  200 100  97-100 Example3 120-240 100-110 71 60-65 In-solvent 120-240 100-110 88 80 method120-240 100-110 84 95 120-240 100-110 100 120-240 100-110 88 84

The results obtained from this NMR study provide strong evidence thatthe solvent-free, stepwise-addition method produces activated PEG withhigher activation degree than the in-solvent method. The results alsoshow that higher activation degree may result if the activator (e.g.p-NPCF) is added in a stepwise manner (c.f. the one-time additionmethod). Also, the reaction time needed to achieve complete activationis generally shorter for the solvent-free method than the in-solventmethod, although the solvent-free method may require a larger amount ofthe activator (e.g., a 67% or higher molar excess).

B. Size Exclusion Chromatography

In addition to NMR spectroscopy, the activation of PEG over time wasmonitored by size exclusion chromatography (SEC).

The samples analyzed by means of NMR were subjected to SEC on a Waterschromatography system consisting of a quaternary pump, an autosamplerand a PDA detector. A TSK 4000 PW_(XL) column coupled with a pre-columnwas used as the separation column.

A mobile phase consisting of 0.1 M ammonium acetate in water was pumpedthrough the column at a flow rate of 1.0 mL/min. The effluent wasdetected at 272 mm, which gives the maximum absorbance signal ofactivated PEG. Injection volume was 20 μL and the elution volume was setto 16 min. To prevent self-decomposition of the samples, the temperatureof the autosampler was set to 12° C.

The PEG-NPC₂ samples were dissolved in water (50 mg/mL) and filteredbefore injection. Each injection was repeated five times for dataverification purposes. Elution curves measured at 272 nm were used aschromatograms for peak area determination.

Preliminary spectrophotometry studies have shown a correlation betweenthe intensity of the absorbance at 272 nm and the activation degree ofPEG-NPC₂. Comparing the areas of the chromatographic peaks recorded at272 nm for samples of known activation degree and those with unknownsubstitution pattern was expected to give a good estimate of theactivation degree of the unknown samples. To confirm the reliability ofSEC as a method for determining the activation degree of a particularPEG-NPC₂ sample, a chromatography analysis was performed with a PEG-NPC₂sample determined to be 100% activated by the NMR technique.

Five injections of this fully activated sample were carried out todetermine the correlation between the peak area of a 100%-activated PEGsample and the activation degree. Using this correlation, the peak areasobtained with 11 subsequent injections of the same sample were used tocalculate the activation degree. Results are summarized in Table 4.

TABLE 4 Calibration values obtained by SEC with 100%-activated PEGsamples. Sample Calculated activation Percentage deviationIdentification degree (%) from nominal value (%) A 94.6 −5.4 B 97.7 −2.3C 99.6 −0.4 D 102.5 2.5 E 100.3 0.3 F 99.3 0.7 G 99.7 0.3 H 101.2 1.2 I104.3 4.3 J 104.4 4.4 K 96.4 −3.6 Mean AD, % = 100 SD(±) = 3.06 SE(±) =0.92

As shown in Table 4, activation degree values calculated from peak areameasurements show a less than 10% deviation from the nominal value. Thisdeviation was deemed to be permissibly small, and SEC was confirmed as areliable method. Accordingly, the activation degree of samples withunknown substitution level was determined from SEC peak areameasurements.

FIG. 6 shows representative chromatograms of activated PEG samplesobtained at 45, 60, 90, and 180 minutes into the reaction. Using peakarea measurements from similar chromatograms, the activation degree ofPEG-NPC₂ samples obtained at different reaction times was determined.The results are graphically displayed in FIG. 4, along with thecalculated values from the NMR study. Both the SEC and the NMR studiessuggest that 100% activation was achieved as early as about 105 minutesinto the reaction and/or with about 66% molar excess of the activatorp-NPCF.

Table 5 compares activation degree values calculated from the SECmeasurements with those calculated with the NMR signals. The activationdegree values obtained by SEC are, in most cases, comparable to thoseobtained by NMR, although the estimated margins of error associated withthe SEC measurements are higher.

TABLE 5 Activation degree values obtained from (i) NMR and (ii) SECexperiments. SAMPLE ACTIVATION ACTIVATION IDENTIFICATION DEGREE (NMR) %DEGREE (SEC) % In-solvent 1 89.5 ± 5.0 95.3 ± 6.9 method 2 68.0 ± 3.074.0 ± 8.1 3 84.0 ± 4.0  63.0 ± 10.0 Solvent-free, 4 95.0 ± 5.0 96.9 ±5.0 stepwise- 5 76.0 ± 4.0 106.7 ± 3.5  addition 6 99.0 ± 1.0  96.3 ±10.9 method 7 82.0 ± 5.0 N/d 8 78.0 ± 1.0 89.1 ± 1.0

C. Melting Points

Melting points of different activated PEG samples were measured using aMeI-Temp® capillary apparatus operated in the temperature range of 20°C. to 60° C. Small amounts of the PEG-NPC₂ samples were placed incapillary tubes and the temperature was raised slowly. The temperatureat which the PEG-NPC₂ powder started to turn into a viscous liquid wasread as the melting temperature. Measurements were taken in triplicates.

FIG. 7 shows the correlation between melting temperatures and activationdegree values. Data were collected from samples prepared by either thesolvent-free stepwise-addition method (Example 2) or the in-solventmethod (Example 3). Samples were collected at different reaction times,their respective activation degrees having been determined previously byeither the NMR and/or the SEC method(s).

It was found that as the activation degree increases, the meltingtemperature decreases. To illustrate, an unsubstituted PEG sample wasfound to melt at about 62.5° C., while a 100%-activated PEG samplemelted at about 52.5° C. This general trend was observed for all studiedsamples, irrespective of the method by which the samples were prepared.Thus the melting point of an activated PEG sample provides a goodestimate of its activation degree.

The inverse correlation between the melting point and the activationdegree of a PEG-NPC₂ sample may be explained by the imperfectionsintroduced into the crystalline structure of the PEG molecules uponsubstitution of the hydroxyl end-groups with nitrophenyl groups. Theseimperfections reduce intramolecular friction between polymer chains andincrease their mobility. Similar decrease in melting points was reportedfor other hydrophobically-modified PEG macromolecules. See Kim et al.,Macromol., 25: 8378-8384 (2002).

EXAMPLE 5 Quantification of Free pNP Content in PEG-NPC₂ Solutions

The amount of free pNP in PEG-NPC₂ samples prepared according to themethod described in Example 2 was determined by high performance liquidchromatography (HPLC). HPLC analysis was performed with an HPLC systemconsisting of a Waters model 600E quaternary solvent delivery system, aWaters model 996 photodiode-array detector, and a Waters 717 plusautosampler. Data analysis was performed with Millennium 32.2 software.The column used was an Ace C4 reversed phase column (15 cm×4.6 mm i.d.)operated at room temperature.

HPLC analyses were carried out in isocratic mode at a flow rate of 2.0mL/min. Elution profiles were analyzed at 317.0 nm. The mobile phase wasa 0.025 M H₃PO₄ solution containing 10% (v/v) of acetonitrile. Beforeperforming the chromatography, the mobile phase was filtered via a 0.22μm filter.

A stock solution of pNP with a concentration of 100 μg/mL was preparedby dissolving a weighted amount of pNP (10 mg) in a 1000-mL volumetricflask. Other standard solutions of pNP with concentrations of 10.0μg/mL, 5.0 μg/mL, 2.5 μg/m L, 1.0 μg/mL, and 0.25 μg/mL were prepared bydiluting the stock 100-μg/mL pNP solution in volumetric flasks.

Weighted amounts of activated PEG samples (500 or 600 mg) were dissolvedin water to achieve 5% (w/v) and 22% (w/v) solutions of the polymer. A20 μL portion of either the 5% or the 22% solution was diluted to atotal volume of 5 mL (dilution factor 250) and the diluted solution wasused for HPLC analysis.

A 25-μL injection of each standard pNP solution and PEG sample solutionwas repeated 5 times on different days to verify intraday and interdayrepeatability of the detector response. The peak area of thechromatogram obtained with each standard solution was plotted againstits known concentration to obtain a calibration curve. The pNP contentin each diluted sample solution was determined accordingly.

The following equation was used to calculate the actual pNPconcentration in each undiluted sample solution:

$\begin{matrix}{{pNP},{{{mg}\text{/}{g({PEG})}} = \frac{{C_{({pNP})}{HPLC}},{{{{\mu g}/{mL}} \cdot 5}\mspace{11mu}{{mL} \cdot 250}\left( {{dilution}\mspace{14mu}{factor}} \right)}}{500\text{,}600\mspace{11mu}{{mg}\left( {{weight}\mspace{14mu}{of}\mspace{14mu}{PEG}} \right)}}}} & \left( {{Equation}\mspace{20mu} 2} \right)\end{matrix}$

The amount of free pNP generated over time in PEG-NPC₂ samples preparedby the solvent-free, stepwise-addition method (Example 2) can be foundin Table 6 below.

TABLE 6 Amount of free pNP generated over time in PEG-NPC₂ samplesprepared by the solvent-free, stepwise-addition method (Example 2). Time(min) pNP (mg/g PEG) SD (± mg/g PEG) 45 15.25 0.11 60 18.21 0.09 7552.51 0.05 90 37.29 0.15 105 37.72 0.36 120 16.68 0.31 180 48.11 3.12

EXAMPLE 6 Purification of the Activated PEG Product

PEG-NPC₂ solutions having concentrations of 5% and 22% (w/v) prepared inExample 6 were mixed with weighted amounts of activated carbon (AC). Theweight ratio of AC/PEG was varied from 0.50% to 50.0%. Suspensions ofactivated carbon in PEG-NPC₂ solutions were incubated for one hour toallow maximum adsorption. Because adsorption reactions are usuallyexothermic, high temperatures are thought to inhibit or slow adsorption.Moreover, an increase in temperature may lead to degradation of theactivated PEG. Accordingly, all sorption-purification procedures werecarried out at room temperature.

After treatment with activated carbon, purified PEG-NPC₂ solutions werefiltered through paper, followed by filtration with 0.22 μm or 0.45 μmmembrane filters, and subjected to lyophilization.

At least two different types of impurities were observed in the PEG-NPC₂samples prepared by the solvent-free method. One of the impurities isfree p-nitrophenol (pNP) generated by side reactions in the moltenPEG-p-NPCF system. The other impurity is believed to bebis-(p-nitrophenyl carbonate)s, a water insoluble material that makesthe PEG-NPC₂ solution prepared by the solvent-free method milky. pNP maybe present as an impurity in the commercially obtained p-NPCF startingmaterial, or it may be a degradation by-product of the activator. It isbelieved that one of the side reactions involves a reaction between thepNP so formed and the starting material p-NPCF. The by-product generatedby this side reaction is believed to be bis-(p-nitrophenyl carbonates).

A small amount of these impurities is known to inhibit desirablereactions between activated PEG and proteins. As many applications ofactivated PEG involve reactions with proteins, an effective method toremove free pNP molecules from the final activated PEG product isneeded.

Activated carbon has been known for a long time as a material with anextraordinarily large surface area and pore volume that gives it aunique adsorption capacity. See Baker et al., Activated Carbon,KIRK-OTHMER ENCYCLOPEDIA OF CHEMICAL TECHNOLOGY, 4:1015-1037 (1992).Another important factor that makes activated carbon an ideal method toremove pNP is that it is a relatively safe substance. Activated carbonis approved by the Food and Drug Administration (FDA) as anover-the-counter drug for humans, and it is not considered a hazardoussubstance by the U.S. Environmental Protection Agency. The use ofactivated carbon to remove pNP is well documented. See Wolborska, Wat.Res., 23:85-91 (1989); Haydar et al., Carbon, 41: 387-395 (2003);Haghseresht et al., Energy & Fuels, 21: 1100-1107 (1998).

It is generally agreed that the aqueous solubility of a solute isinversely related to its adsorption on carbon. As expected, thewater-insoluble material observed in aqueous solutions of PEG-NPC₂ waseffectively removed by activated carbon.

Table 7 summarizes the amount of pNP in PEG-NPC₂ solutions before andafter purification with activated carbon.

TABLE 7 Efficiency of activated carbon (AC) in removing pNP. A 1:1weight ratio of AC/PEG was used, i.e., 250 mg of activated carbon wasadded to every 250 mg of activated PEG. Initial concentrationPost-treatment concentration Reaction pNP SD PNP SD Efficiency of time(min) (mg/g PEG) (±mg/g PEG) (mg/g PEG) (±mg/g PEG) sorption (%) 4515.25 0.11 0.60 0.001 96.05 60 18.21 0.09 0.89 0.005 95.09 75 52.51 0.052.96 0.008 94.37 90 37.29 0.15 2.73 0.005 92.69 105 37.72 0.36 1.820.022 95.16 120 16.68 0.31 n/d n/d 180 48.11 3.12 1.82 0.007 96.22 Mean94.93 Efficiency (%)

The efficiency of activated carbon in removing pNP was calculated by thefollowing equation:

$\begin{matrix}{{Efficiency},{\% = {{\frac{{pNP}_{Initial} - {pNP}_{{after}\mspace{14mu}{treatment}}}{{pNP}_{Initial}} \cdot 100}\%}}} & \left( {{Equation}\mspace{20mu} 3} \right)\end{matrix}$

As seen from the results summarized in Table 7, a 1:1 weight ratio ofactivated carbon to PEG-NPC₂ had proven to be highly efficient inremoving residual pNP. The efficiency of purification was observed to beabout 95% for all studied concentrations of pNP (15-48 mg/g) in PEG-NPC₂solutions, suggesting that a smaller amount of activated carbon may beused and still be effective.

Additional experiments were performed on 22% (w/v) activated PEGsolutions to determine the optimal AC/PEG ratio for removing 90-95% offree pNP. To these solutions were added the following amounts ofactivated carbon: 0.5, 2.5, 5.0, 10.0, 20.0 and 50.0% (w/w) (Table 8).The resulting mixture was continuously stirred for an hour to allowmaximum adsorption.

TABLE 8 Activated carbon/PEG-NPC₂ weight ratio used to purify PEG-NPC₂.PEG CONCEN- Activated TRATION, Weight of carbon Act. Carbon/PEG (mg/mL)polymer (mg) added (mg) ratio (%) 220 1100 5.5 0.5 220 1100 27.5 2.5 2201100 55.0 5.0 220 1100 110.0 10.0 220 1100 220.0 20.0 220 1100 550.050.0

HPLC analysis was performed to quantify the pNP level in PEG-NPC₂samples before and after treatment with activated carbon. Procedureswere described in Example 6. The efficiency of the treatment wasdetermined by Equation 3, and the results are graphically displayed inFIG. 8.

It was found that the efficiency of sorption increases with the amountof activated carbon added. pNP was 100% removed with a 50% weight ratioof AC/PEG-NPC₂. However, it can be seen from FIG. 8 that even a 5-10%weight ratio of activated carbon was sufficient to remove up to 90-95%of pNP.

It is known that the adsorption amount is dependent upon theconcentration of the adsorbent, the temperature of the solution, and thepolarity of the adsorbent.

Usually, the isotherm of activated carbon sorption is described by theFreundlich function given below:

$\begin{matrix}{{\frac{{pNP}({mg})}{{AC}({mg})} = {K_{f}C_{e}^{\frac{1}{n}}}},} & \left( {{Equation}\mspace{20mu} 4} \right)\end{matrix}$where pNP (mg)/AC (mg) is the amount of pNP adsorbed per milligram ofactivated carbon, C_(e) is the concentration of pNP at equilibrium afterabsorption, K_(f) is an absorbent capacity constant, and n is acharacteristic constant of the absorbent surface. See Ko et al., Carbon,40, 14: 2661-2672 (2002).

Data from Table 8 were substituted into Equation 4. The correspondingplot was given in FIG. 9. A linear correlation was observed whensorption capacity (pNP (mg)/AC (mg)) was plotted against C_(e) in doublelog coordinates in an AC concentration range of 27.5-550 mg/mL. Theparameter K_(f) was determined to be K_(f)=0.01 mL/mg and n=0.493,indicating good realization of sorption properties of activated carbonwhen applied to a relatively concentrated PEG-NPC₂ solution. TheFreundlich function, however, gives very little information about theoptimal concentration of activated carbon to be used.

To estimate the optimal range of AC concentration, the followingequation is used to calculate a specific sorption parameter:

$\begin{matrix}{{Specific}\mspace{14mu}{sorption}\text{:}\mspace{11mu}\frac{{\mathbb{d}{pNP}}\mspace{14mu}{{absorbed}({mg})}}{{\mathbb{d}{AC}}\mspace{14mu}{{added}({mg})}}} & \left( {{Equation}\mspace{20mu} 5} \right)\end{matrix}$The specific sorption value given by Equation 5 indicates the increasedamount of pNP absorbed per mg of carbon added. The calculated specificsorption values are plotted against the weight ratio of AC/PEG in FIG.10. The optimal amount of activated carbon to be added per mg ofPEG-NPC₂ to remove 90-95% of free pNP corresponds to the sharpest curvein the plot, which was found to be between 10-20% (w/w) of AC/PEG.

The impurities mentioned above also may be removed by means ofion-exchange, followed by filtration of the purified polymer solutionsthrough 0.22 μm membrane filter. Ion-exchange resins are known to be anefficient means to absorb nitrophenolic compounds from aqueousenvironment (see Ku et al., J. Hazard. Mater., 80: 59-68 (2000); Li etal., J. Environ. Sci. (China), 14: 457-463 (2002); Abburi et al., J.Hazard. Mater., 105: 143-156 (2003); and Calace et al., Environ.Pollut., 118: 315-319 (2002)), whereas microfiltration helps to removeinsoluble particles of bis-(p-nitrophenyl carbonates).

Analysis of pNP in the PEG-NPC₂ samples prepared and purified accordingto the method described in Example 5C, shows that the amount of pNPdecreases significantly upon deionization of the polymer solution viaion-exchange treatment. Table 9 summarizes these results.

TABLE 9 Amount of pNP in PEG-NPC₂ samples prepared according to themethod described in Example 5C. Amount of pNP before Amount of pNP afterPEG-NPC2 ion-exchange ion-exchange Samples (mg/g) ± SD (mg/g) (mg/g) ±SD (mg/g) A 19.44 ± 0.05 10.60 ± 0.08 B 19.44 ± 0.05 12.71 ± 0.01 C17.33 ± 0.15 n/d

Both the ion-exchange and active carbon treatments are by far lessexpensive and less polluting than traditional techniques using organicsolvents to recover and purify polymer (see, e.g., Example 3). To obtainhighly purified activated PEG, one may treat raw activated PEG productsfirst with ion-exchange resins, followed by further purification withactivated carbon.

EXAMPLE 8 Lyophilization of Purified PEG Solutions

Clear colourless or slightly yellowish solutions of PEG-NPC₂ with aconcentration of 22% (w/v) or 30% (w/v), prepared according to theprocedures described in Example 6, were transferred into freeze-dryerflasks. The solutions were cooled to −40° C. in rotatory shell freezerfilled with 70% ethanol in water. The freeze-dryer flasks containing thefrozen PEG solutions were then connected to the vacuum lines of afreeze-dryer (Labconco) connected to a condensation camera stabilized at−40° C. to −50° C. The frozen PEG solutions were dried for 24 to 48hours.

EXAMPLE 9 Influence of Purification and Neutralization Techniques onActivation Degree of Activated PEG

A. Preparation of PEG-NPC₂

PEG-8000 (Fisher Scientific, 1000 g, 125 mmol) was placed in a reactorequipped with a thermometer and a mechanical stirrer. The reactionsystem was thermostabilized at 70° C. Vacuum was applied upon continuousstirring to remove air from the system. Once the PEG powder wascompletely melted, p-NPCF (ABCR GmbH & Co. KG, Karlsruhe, Germany, 15.0g, 74.5 mmol) was added to the molten PEG. The reaction mixture wasincubated under vacuum for 30 minutes. More p-NPCF was added in 15-gramportions at 30-min intervals until a 60% molar excess of p-NPCF had beenadded (i.e., 75 g of p-NPCF per 1000 g of PEG). The reaction mixture wasstirred at 70° C. for two hours to remove residual HCl vapors. ThePEG-NPC₂ melt was transferred into 4.5 L of cold distilled water, anddissolved to provide a 22 wt. % aqueous solution. The resultant aqueoussolution was divided into 3 samples and subjected to three differentpurification methods as detailed below.

B. Purification with Activated Carbon

Activated carbon (33 g, 10 wt. % of PEG-NPC₂) was added to a firstsample of the PEG-NPC₂ aqueous solution obtained from Part A. Themixture was filtered and lyophilized to provide purified non-neutralizedPEG-NPC₂.

C. Purification with Anion-exchange Resin

A second sample of the PEG-NPC₂ aqueous solution obtained from Part Awas treated with anion-exchange resin (Amberlite® IRA-67, 33 g, 10 wt. %of PEG-NPC₂) to remove residual HCl. Once the pH of the activated PEGsolution reached 5.0, the solution was filtered and freeze-dried to givepurified deionized PEG-NPC₂.

D. Neutralization with NaOH

An aqueous solution of 6N NaOH was added to a third sample of thePEG-NPC₂ aqueous solution obtained from Part A to adjust the pH value to5.5. The solution was filtered and dried to give purified neutralizedPEG-NPC₂.

E. Activation Degree as Determined by SEC

It was found that different purification and neutralization techniquescould affect the activation degree of the final activated PEG product.Table 10 compares the activation degree of PEG-NPC₂ samples purifiedand/or neutralized according to the three techniques described in PartsB-D above, as determined by SEC.

TABLE 10 Influence of purification/neutralization technique onactivation degree. Activation degree pH value of Deviation from initialPEG-NPC₂ 22% PEG-NPC₂ value before purification/ Samples solutionAverage (%) neutralization (%) Purified non- 2.3 97.76 0 neutralized(Part B) Purified 5.2 95.24 −2.6 deionized (Part C) Purified 5.7 86.08−11.95 neutralized (Part D)

As shown by the results in Table 10, when an activated PEG sample wasneutralized with NaOH, its activation degree decreased. This can beexplained by the effect of local hydrolysis of the carbonate linkages inat least some of the PEG-NPC₂ molecules by the strong base NaOH.

In contrast, the ion-exchange treatment proceeds under much milderconditions, thus helping to preserve the initial degree of substitution.The results in Table 10 show that only 2.6% of the substitution was lostupon neutralization via ion exchange. The resultant polymer is referredto as purified deionized activated PEG, since C1⁻ ions from HCl arereplaced by OH⁻ ions upon ion exchange, resulting in the formation ofwater molecules.

EXAMPLE 10 Synthesis of PEG-protein Hydrogel.

PEG-NPC₂ (5.5 g) prepared according to the method described in Example 2were added to 25 mL of deionized water. Hydrolyzed soy protein, casein,and soy albumin, respectively, was dissolved in 0.14M NaOH to give 12%(w/v) (120 mg/mL) protein solutions. The pH of the protein solutions wasadjusted to 11.80. The polymer solutions were mixed with equal volume ofone of the protein solutions, and the mixture was placed between twopieces of glass to form a gel with a thickness of 1.8 mm. The resultinghydrogel samples (PEG-protein hydrogels) were washed in EDTA/NaCl bufferto remove residual pNP.

Completely swollen hydrogel samples were observed to be a transparent toopaque rubbery material. No visible differences were observed betweensamples obtained with solvent-free activated PEG (i.e., preparedaccording to the procedures described in Example 2) and those obtainedwith in-solvent activated PEG (i.e., prepared according to theprocedures described in Example 3).

Compression tests were performed to compare the mechanical properties ofhydrogels prepared by solvent-free activated PEG and in-solventactivated PEG. The results suggested that the mechanical properties ofhydrogels made with either solvent-free or in-solvent activated PEGs aresimilar when the hydrogels were prepared with the same proteinsolutions. Other physicochemical properties of the hydrogels, such astheir equilibrium water content, also seem to be unaffected by theprocedure by which the activated PEG was prepared.

Variations, modifications, and other implementations of what isdescribed herein will occur to those of ordinary skill in the artwithout departing from the spirit and the essential characteristics ofthe invention. Accordingly, the scope of the invention is to be definednot by the preceding illustrative description but instead by thefollowing claims, and all changes that come within the meaning and rangeof equivalency of the claims are intended to be embraced therein.

Each of the patent documents and scientific publications disclosedhereinabove is incorporated by reference herein for all purposes.

1. A method for preparing an activated polyethylene glycol having theformula:Y-Q-(OCH₂CH₂)_(n)—O-Q-Y wherein Q is —C(O)—; Y is a leaving group; and nis an integer greater than 2; the method comprising: heating apolyethylene glycol to provide a molten polyethylene glycol; andreacting the molten polyethylene glycol with an activator having theformula:Y-Q-X wherein X is a leaving group; and Q and Y are as defined above;wherein the reaction is carried out in the absence of a solvent and inthe absence of a base.
 2. The method of claim 1 wherein Y is selectedfrom a halide group, a mesyl group, a tosyl group, a phenoxyl group, anda substituted phenoxyl group.
 3. The method of claim 2 wherein X isselected from a halide group, a mesyl group, a tosyl group, a phenoxylgroup, and a substituted phenoxyl group.
 4. The method of claim 1wherein the activator is O₂NPhOC(O)Cl.
 5. The method of claim 1 whereinthe reacting step is carried out at a temperature at least about 10° C.above the melting point of polyethylene glycol.
 6. The method of claim 1wherein the polyethylene glycol has a molecular weight between about6,000 and about 11,000 Da, and the reacting step is carried out at atemperature in the range of about 60-90° C.
 7. The method of claim 1wherein the reacting step is carried out in an inert atmosphere.
 8. Themethod of claim 1 wherein the molar amount of the activator is in excessof the molar amount of the molten polyethylene glycol.
 9. The method ofclaim 1 wherein the molar excess is between about 50-100%.
 10. Themethod of claim 1, wherein the molten polyethylene glycol is reactedwith the activator for less than or about two hours.
 11. The method ofclaim 1, wherein the activated polyethylene glycol has an activationdegree of greater than about 90%.
 12. The method of claim 1 comprisingcontacting the activated polyethylene glycol with ion exchange resins.13. The method of claim 1, wherein the polyethylene glycol has theformula:M-(OCH₂CH₂)_(n)—O-M, and wherein M is selected from H, Li, Na, K, Rb,and Cs.
 14. A method for preparing an activated polyethylene glycol, themethod comprising: heating a polyethylene glycol to provide a moltenpolyethylene glycol; and reacting the molten polyethylene glycol withO₂NPhOC(O)Cl to provide an activated polyethylene glycol comprising atleast one terminal group of O₂NPhOC(O)—; wherein the reaction is carriedin the absence of a solvent and in the absence of a base.